Protein Protocols Protein Protocols

Casting IPGs 171
pH gradients, whereas migration on continuous or gradient PAA slabs of different %T
in 2-D may further improve resolution of the spots of interest.
In order to optimize reproducibility and to increase throughput, laboratories handling
a very large number of gels may consider a medium-scale production with gradient
pouring through mechanical devices (computer-driven burettes) (8,9). In any event,
even at the typical laboratory level, the procedure of IPG casting is reliable and allows
a remarkable reproducibility of results (10). The following will try to convince the
readers that it is also an easy one.
2. Materials
2.1. Equipment
1. Polymerization cassette:
a. A molding plate with a 0.5-mm-thick permanent frame. Available from Pharmacia in
either 12.5 × 25 cm, w × h (cat. no. 18-1013-74), or 25 × 12.5 cm, w × h, size (cat. no.
80-1106-89).
b. Gel-supporting plate.
c. Gel-binding foil, e.g., GelBond PAG (Pharmacia).
d. Clamps.
2. Gradient-mixing device:
a. A two-vessel chamber (e.g., gradient maker from Pharmacia, cat. no. 18-1013-72).
b. Magnetic bars.
c. Outlet to the mold, either a one-way silicone tubing (od 3 mm, id 1.5 mm, ca. 12 cm
long, equipped with a 2-cm teflon tip) or multiple inlets cast by adapting the tips, ca. 6
cm long, of butterfly needles gauge 21 to a T-connector, available from Cole-Parmer
(cat. no. K6365-70).
d. A stirrer providing constant and even operation at medium to low rpm rating with
minimal overheating.
e. Screw-jack rising table.
3. Forced-ventilation oven at 50°C.
4. Shaking platform.
5. Fan.
2.2. Reagents
1. Acrylamide buffers: 0.2-M solutions (see Table 1), prepared either in water with 5 ppm of
hydroquinone methylether as polymerization inhibitor for the acidic monomers or in
n-propanol for the basic monomers. The chemicals are available from Pharmacia (as
Immobiline) or from Fluka (Buchs, Switzerland). Storage at 4°C; the expiration date is
given by the manufacturer. The products are defined as irritant, whereas n-propanol is
classified as highly flammable.
2. Acrylamide monomers stock solution: 30% T, 4% C (11). Dissolve in water 1.2 g bisacrylamide
and 28.8 g acrylamide/100 mL final volume. Filter the stock through
0.8-mm membranes and store at 4°C for about 2 mo. The chemicals should be of the
highest purity. Acrylamide has been recognized as a neurotoxin.
3. 40% w/v Ammonium persulfate solution: Dissolve 200 mg of ammonium persulfate in
440 µL of water. Store up to a week at 4°C.
4. TEMED: Store at 4°C for several months.
5. 87% v/v Glycerol.